毛细管在酶分析和免疫分析方面的研究及应用

发布时间：2018-01-02 23:25

本文关键词：毛细管在酶分析和免疫分析方面的研究及应用　出处：《东北师范大学》2017年博士论文　论文类型：学位论文

【摘要】：毛细管由于传质距离短、试剂消耗量少、便于携带、使用简便等优势,现已广泛应用于分析化学中的各个方面。本论文围绕熔融石英毛细管在酶的在线连续监测及构建免疫传感器方面开展了以下研究工作:1.基于毛细管电泳(CE)技术,成功构建了酶反应在线分析体系,用于监测酶反应过程。利用穿过样品池的相距15μm的两根同轴毛细管,成功构建了毛细管电泳连续分析的装置,该装置设计简单,操作方便,通过在样品池中施加额外的电压,实现了样品的在线连续进样和毛细管电泳分析。结合在线快速衍生和毛细管电泳技术,通过在线连续监测脲酶产物(氨)的生成,对脲酶的活性进行了研究。脲酶反应过程中,只有产物氨可以与衍生试剂发生反应,并在紫外区(340 nm)有吸收,谱图斜率即产物生成速率,也是酶反应速率。所得米氏常数Km和最大反应速率Vmax分别为2.79±0.15 mM和4.32±0.20 mM/min(n=3),与传统线外方法所得结果一致,表明本方法对于在线酶分析测定具有很好的准确性和可行性。基于此方法研究了Cu2+的抑制常数和机理。根据Km和Vmax值的变化规律,得知Cu2+为非竞争型抑制剂,抑制常数Ki为0.34±0.02μM,该结果与线外测定结果相符,证实了该方法在研究酶抑制动力学方面的准确性。此研究对于疾病治疗中控制脲酶活性、农业上控制土壤中氮肥具有潜在应用价值。2.提出了将光门进样与紫外/可见检测相结合的(OG-UV/vis)序列毛细管电泳分析,实现了酶反应的全程在线监测。光门进样是一种快速、连续、可重复的进样方式,样品池中的酶反应产物连续的进入毛细管,在UV/vis检测之前用相应波长的激光束照射一段时间使样品的紫外吸收减弱,在谱图中形成负峰。实验详细考察了漂白光强度(激光器功率)、光门进样时间、运行电压、衍生试剂浓度对灵敏度及分离的影响。以L-天冬酰胺酶为例,通过同时测定底物的消耗和产物的生成,实现了酶催化反应线上全程监测,时间分辨率为12s,即每隔12s就可获得一组底物、产物峰,Km和Vmax值分别为0.25 mM和0.24 mM/min,与传统线外方法一致,表明了OG-UV/vis的可行性。该方法仪器简单,分析快速,自动化高,作为连续在线过程分析的有力工具,可用于在线监测各种生物进程。3.制备了多通道微结构毛细管免疫传感器,并对其性能进行了研究。实验采用双抗体夹心法,通过化学键合法将抗体固定于毛细管内壁,免疫反应完成后,用激光诱导荧光技术(LIF)进行检测。与相同截面积的普通毛细管相比,其表面积的增加有利于分子固定和识别,使得灵敏度提高了大约5倍。此外,我们也对封闭剂和包被抗体(Ab1)的浓度进行了优化,并在最佳条件下考察了传感器的批内、批间重现性,分别为4.43%和7.14%(n=3),说明该方法具有较好的重现性。4.多通道微结构毛细管荧光免疫传感器对肿瘤标志物的检测。以检测原发性肝癌的特异性标志物-甲胎蛋白(AFP)为例,首先绘制了AFP的标准曲线,所得线性范围、检出限与传统ELISA方法进行比较。在最佳条件下考察了传感器的特异性,实验结果表明,所制备的荧光免疫传感器具有强的抗干扰能力,适用于复杂样品的分析。实验分别测定了单一血清样品的回收率和多个临床样品中的AFP含量,回收率在84.3%-119.0%之间,所得AFP浓度与临床检测结果进行比较,相对误差小于20.73%,相关性方程为y=0.888x+0.056。通过更换抗原、抗体体系,所制备的PCF免疫传感器有望应用于对其他肿瘤标志物的检测,在临床分析中具有潜在的应用价值。
[Abstract]:Because the capillary mass transfer distance is short, less reagent consumption, easy to carry, easy to use and other advantages, has been widely used in various aspects of chemical analysis. This paper focuses on aspects of fused silica capillary enzyme in online continuous monitoring and construction of immune sensor to carry out the following research work: 1. based on capillary electrophoresis (CE) technique, was successfully constructed the analysis system for online monitoring of enzyme reaction, enzyme reaction process. Through the use of sample pool is 15 mu m two coaxial capillary, successfully constructed device for continuous analysis of capillary electrophoresis, the device has the advantages of simple design, convenient operation, by applying additional voltage in the sample pool, realizes on-line continuous sampling and sample capillary electrophoresis analysis. Combined with the fast online derivative and capillary electrophoresis technology, products through online continuous monitoring of urease (ammonia) generation of urease activity were studied. The urease reaction process, only the product can react with ammonia and derivatization reagent in the ultraviolet region (340 nm) absorption spectra, the slope of that product formation rate, and enzyme reaction rate. The Michaelis constant Km and the maximum reaction rate of Vmax were 2.79 + 0.15 and 4.32 + 0.20 mM mM /min (n=3). With the traditional line method have the same results show that this method has good feasibility and accuracy of the determination of the analysis for the online enzyme. Based on this method to study the inhibition constants and the mechanism of Cu2+. According to the variation of Km and Vmax value, that Cu2+ as a non competitive inhibitor, the inhibition constant Ki was 0.34 + 0.02 M the results, and line determination results, confirmed the accuracy of this method in the study of enzyme inhibition kinetics. The research for the control of urease activity in the treatment of diseases, control of soil nitrogen in agriculture has the potential application value of.2. is proposed to be light The door of sampling and ultraviolet / visible detection combination (OG-UV/vis) sequence analysis of capillary electrophoresis, realized the on-line monitoring of the whole enzyme reaction. Optical gate injection is a rapid, continuous, sample into repeat, into the capillary enzyme reaction products in the sample cell continuous, UV to sample the corresponding wavelength of the laser beam irradiation time in UV/vis detection before the decrease of absorption, formation of negative peaks in the spectrum. The experiment investigated bleaching light intensity (laser power), light door operation voltage, injection time, derivatization reagent concentration separation effect on sensitivity and to L- asparaginase as an example, by generating simultaneous determination of substrate consumption and product, the enzyme catalytic reaction online monitoring, time resolution of 12s, is that every 12s can get a set of substrate, product peak, Km and Vmax were 0.25 mM and 0.24 mM/ min, and the traditional line method Consistent, show that OG-UV/vis is feasible. The method is simple, rapid analysis, high automation, as a powerful tool for continuous online analysis process, can be used for online monitoring of various biological processes for preparation of.3. multi-channel micro structure capillary immunosensor, and its performance was studied. The experiment using double antibody sandwich method by chemical bonding will be fixed on the inner wall of the capillary, antibody, immune response after using laser induced fluorescence technique (LIF) were detected. Compared with the same cross-sectional area of the common capillary, its surface area increased with the increasing of molecular recognition and fixed, the sensitivity increased by about 5 times. In addition, we also on sealant and coating antibody (Ab1) concentration were optimized, and the optimum conditions were investigated in batch batch sensor, reproducibility, respectively 4.43% and 7.14% (n=3), which shows that the new method has a good reproducibility of.4. Multi channel micro capillary fluorescence immunosensor for the detection of tumor markers. The specific marker detection of primary liver cancer alpha fetoprotein (AFP) as an example, first draw the standard curve of AFP, the linear range, detection limit and traditional ELISA method were compared. The optimum conditions were investigated specific the sensor, the experimental results show that the fluorescence immunosensor prepared has strong anti-interference ability, suitable for the analysis of complex samples. The experiment tested the content of AFP recovery single serum samples and multiple clinical samples, the recovery rate was 84.3%-119.0%, compared with AFP concentration and clinical detection results, the relative error is less than 20.73%, the correlation equation was y=0.888x+0.056. by replacing the antigen antibody system for PCF immune sensor preparation is expected to be applied to other tumor markers in clinical detection. The analysis has potential application value.

【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O658.9;TP212.3

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